1. Field of the Invention
This invention relates to certain derivatives of flavin adenine dinucleotide (FAD) which are useful as intermediates in the synthesis of FAD-labeled conjugates used as reagents in specific binding assays.
Flavin adenine dinucleotide has the following chemical structure [The Merck Index, 9th ed. (1976) p. 532]: ##STR2## which hereinafter is abbreviated as: ##STR3## wherein Riboflavin--Phos).sub.2 Ribose represents the riboflavin-pyrophosphate-ribose residue in FAD.
2. Description of the Prior Art
Specific binding assay methods have undergone a technological evolution from the original competitive binding radioimmunoassay (RIA) in which a radioisotope-labeled antigen is made to compete with antigen from a test sample for binding to specific antibody. In the RIA technique, sample antigen is quantitated by measuring the proportion of radioactivity which becomes associated with the antibody by binding of the radiolabeled antigen (the bound-species of the labeled antigen) to the radioactivity that remains unassociated from antibody (the free-species) and then comparing that proportion to a standard curve. A comprehensive review of the RIA technique is provided by Skelly et al, Clin. Chem. 19:146(1973). While by definition RIA is based on the binding of specific antibody with an antigen or hapten, radiolabeled binding assays have been developed based on other specific binding interactions, such as between hormones and their binding proteins. All radiolabeled specific binding assays are by necessity heterogeneous, that is, the bound- and free-species of the labeled conjugate must be physically separated and the label (i.e., radioactivity) measured in one of the separated species.
From the radiolabeled binding assays have evolved nonradioisotopic binding assays employing labeling substances such as enzymes as described in U.S. Pat. Nos. 3,654,090 and 3,817,837. Recently, further improved nonradioisotopic binding assays have been developed as described in German Offenlegungschriften Nos. 2,618,419 and 2,618,511, based on U.S. Ser. Nos. 667,982 and 667,996, filed on Mar. 18, 1976 and assigned to the present assignee, employing particularly unique labeling substances, including coenzymes, cyclic reactants, cleavable enzyme substrates, and chemiluminescent molecules. Flavin adenine dinucleotide (FAD) is mentioned as being useful as a coenzyme label since FAD functions as a coenzyme in reactions which can be used to monitor specific binding reactions. The majority of the recently developed nonradioisotopic specific binding assays can be performed in a homogeneous format, that is, without separating the bound- and free-species of the labeled conjugate, due to the fact that the label expresses a different activity in the bound-species compared to the free-species. In particular, FAD has been found to be useful as a prosthetic group label as described in U.S. patent applications Ser. Nos. 917,961 and 45,423, filed June 22, 1978 and June 4, 1979, respectively, both assigned to the present assignee. U.S. Pat. No. 4,171,432 issued to the present assignee and U.S. patent application Ser. No. 950,858, filed Oct. 12, 1978 and assigned to the present assignee, describe certain FAD-labeled conjugates and intermediates in the synthesis thereof.
Modification of FAD and related adenine derivatives is described in Eur. J. Biochem 89:491(1978) and U.S. Pat. No. 4,008,363.